There is a fundamental gap in our understanding of the mechanism by which the highly pathogenic ebolaviruses (EBOV) and marburgviruses (MARV) enter their host cells and initiate infection. Our long-term objectives are to understand, in molecular detail, how these viruses exploit their host cells to gain entry into the cytoplasm, and to contribute to the development of antiviral therapies for EBOV/MARV infection. The focus of this particular application is the mechanism by which endosomal cysteine proteases, a class of cellular enzymes we identified to be essential EBOV/MARV entry host factors, activate GP to fuse viral and cellular bilayers. Our central hypothesis is that the proteolytic disassembly of GP within host cell endosomes, acting either alone or in concert with additional cellular stimuli, provides the trigger signal that releases GP from its pre-fusion conformation and induces viral membrane fusion. Guided by strong preliminary data, we will test this hypothesis by pursuing two specific aims: 1. Identify the critical molecular features of GP proteolytic disassembly by endosomal cysteine proteases during EBOV and MARV entry 2. Reconstitute viral membrane fusion in vitro and elucidate the roles of proteases and non-protease host factors in fusion triggering PUBLIC HEALTH RELEVANCE: Ebolaviruses and marburgviruses are the cause of an invariably fatal hemorrhagic fever in humans in equatorial Africa, and are also potential agents of bioterrorism. We expect this work to provide information crucial to the design and development of anti-EBOV/MARV therapies targeting cellular cysteine proteases. We also expect to find new targets, within the virus or the host cell, for developing antiviral drugs.